Cholinergic
and VIP-ergic pathways mediate histamine
Hz
receptor-induced
cyclical secretion in the guinea pig colon
HELEN
J. COOKE, Y.-Z. WANG, RHODA REDDIX,
AND NAJMA JAVED
Department
of Physiology,
College of Medicine, The Ohio State University,
Columbus,
Cooke,
Helen
J., Y.-Z. Wang, Rhoda
Reddix,
and
Najma Javed. Choline@
and VIP-ergic pathways mediate
histamine Hz receptor-induced
cyclical secretion in the guinea
pig colon. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31):
G465-G470,
1995.-Previous
studies demonstrated
neurally
mediated recurrent
increases in short-circuit
current
(I,,)
suggestive of anion secretion in guinea pig distal colon. To
determine the neural pathways involved, segments of distal
colon from guinea pigs were mounted in flux chambers. In
muscle-stripped
or whole thickness preparations,
serosal addition of the histamine
Hz receptor agonist, dimaprit,
caused
cyclical increases in I,,, which were reduced by the chloride
channel blocker, N-phenylanthranilic
acid, but not by the
sodium channel blocker amiloride.
Dimaprit
stimulated
release of [3H1acetylcholine
and vasoactive intestinal polypeptide
(VIP) from submucosalimucosal
sheets. Dimaprit caused recurrent increases in I,,, which were significantly
decreased by
mecamylamine,
a nicotinic
receptor antagonist,
and nearly
abolished
by
the
muscarinic
antagonist,
atropine
antagonist,
4-diphenylace043
> Ml = Mz). The muscarinic
toxy-N-methyl-piperidine
methiodide
(4-DAMP,
M3 > Ml),
was more potent than pirenzepine
(M1 > M3) in reducing
recurrent
increases in I,,. Dimapritand electrically
evoked
secretion were inhibited
by the VIP antagonists
[4Cl-D-Phe6,
Leu17]VIP and VIP hybrid. The results suggest the involvement of VIP-ergic and cholinergic
neurons utilizing
nicotinic
and muscarinic synapses in mediating secretion.
submucosal
plexus; chloride
secretion;
dimaprit;
HZ receptor
WHICH
IS RELEASED
from mast cells by immunologic and nonimmunologic stimuli, is an important
mediator of gastrointestinal function (16, 31). Histamine stimulates gastric acid and intestinal sodium and
chloride secretion, inhibits sodium and chloride absorption, relaxes or contracts smooth muscle cells, and
increases blood flow (1, 6, 7, 22, 27, 30, 32). Histamine
has been reported to be the cause of watery diarrhea
syndrome in some patients with microscopic colitis and
to contribute to diarrhea during intestinal allergic reactions (2). There are multiple target sites for histamine’s
action that could explain its ability to alter salt and
water transport. Besides its well-known effect on epithelial cells to stimulate chloride secretion or alter sodium
and chloride transport via its interaction with H1 receptors, it affects other target cells to release prostaglandins, which are well-known secretagogues (14, 27, 30,
32) .
Another important site of action of histamine is on
enteric neurons. Histamine’s immediate effects are depolarization of submucosal neurons and enhanced neuronal excitability (8). Prolonged exposure in the guinea pig
colon to histamine or the Hz receptor analogue, dimaprit, leads to a bursting pattern of neuronal firing,
which is the driving force for recurrent increases in
HISTAMINE,
0193~1857/95
$3.00
Copyright
Ohio 43210
short-circuit current (ZSc)indicative of chloride secretion
(8, 29). The ability of tetrodotoxin and cimetidine to
abolish completely the cyclical changes in I,, suggests
that histamine acts at Hz receptors to modulate neural
activity (8,29). The neuronal pathways mediating histamine Hz activation and recurrent increases in Z,, are
unknown. Nevertheless, cholinergic, VIP-e@, and substance P-containing neurons have been implicated in
secretory reflexes in the intestine and therefore are
potential sites of histamine’s action (3, 4, 9-12, 19, 25).
The aim of this study was to determine whether
choline@
or VIP-ergic neurons mediate dimapritevoked recurrent increases in I,,. The results suggest
that activation of histamine Hz receptors increases Z,,
and chloride secretion by stimulating choline@
neurons that utilize muscarinic and nicotinic synapses and
by activating VIP-e@ pathways.
METHODS
Tissue preparation.
Male Hartley guinea pigs (Harlan Sprague Dawley, Indianapolis,
IN), weighing
350-500
g, were
stunned and exsanguinated.
Segments of distal colon, 10 cm
proximal
to the anus, were excised and opened along the
mesenteric border. The intraluminal
contents were removed
and whole thickness
or muscle-stripped
segments
were
mounted
in flux chambers
for measurement
of electrical
parameters
(21, 30). The serosal surface area was 0.78 cm2,
Tissues were bathed on both sides with 5-10 ml of KrebsRinger solution warmed to 37°C and bubbled with 95% 02--5%
C02. Unless otherwise
stated, the solution consisted of the
following
(in mM): 120 NaCl, 6 KCl, 1.2 MgC12*6 HzO, 1.3
NaH2P04*H20,
14.4 NaHC03, 2.5 CaC12, and 12.5 glucose.
The solutions were buffered at N pH 7.2.
ELectricaL measurements.
The flux chambers were equipped
with Ringer-agar
bridges and calomel half cells for measurement of transmural
potential
difference. A short-circuiting
current was passed from a voltage-clamp apparatus (Physiological Instruments,
VC-600, Houston, TX) via silver-silver
chloride electrodes to abolish the transepithelial
potential difference (21, 30). Submucosal neurons were stimulated via a pair
of aluminum
foil electrodes juxtaposed
to the submucosal
surface of the tissue. The electrodes were connected to a Grass
SD-9 stimulator
(Grass Instruments,
Quincy, MA), which
generated
stimulus
pulses of O.5-ms duration,
lo-35
V
strength, and IO-Hz frequency. Changes in I,, were continuously monitored
on a chart recorder or IBM-XT
computer.
Measurements
of the changes in I,, during either electrical or
chemical stimulation
were determined
as the difference between the peak response and baseline I,, before stimulation.
Dimaprit-evoked
recurrent increases in ISC.Previous studies
showed that the recurrent
increases in I,, caused by dimaprit
were all or none and occurred at a threshold concentration
of 1
or 2 PM (29). Higher concentrations
up to 110 PM evoked the
same amplitude response (29). Suprathreshold
concentrations
of dimaprit (2.5,5, or 10 FM) were added to the serosal bath to
ensure an effect of dimaprit.
If recurrent
increases in I,, did
not occur with the first concentration
within 15 min, a second
o 1995 the American
Physiological
Society
G465
G466
H2 RECEPTOR-EVOKED
concentration
was added as described previously (29). Drugs
were usually added after the third of fourth recurrent cycle.
AcetyZchoZine reZease studies. The submucosal
surfaces of
muscle-stripped
tissues were bathed for 30 min at 37°C with 4
ml Krebs-Ringer
buffer containing
1 FM choline chloride and
10 @i/ml (86.7 Ci/mmol, New England Nuclear) [3H]choline.
During the uptake period, submucosal neurons were continuously stimulated
at 1 Hz, 35 V, and 2 ms for 30 min (17, 18).
This was followed by another 30-min incubation
period without electrical stimulation.
The fluid was discarded, and the
submucosal
surface was perfused at 1 ml/min with KrebsRinger solution
with 10 C_LM hemicholinium-3
and 1 PM
choline chloride
for 60 min. One-milliliter
samples were
collected continuously
every minute. Five samples were collected to establish basal release followed by collection of an
additional
38 samples in the presence of 10 PM dimaprit
as
previously described (17, 18).
The outflow of 3H from colonic segments preloaded with
C3H]choline was measured in the absence of cholinesterase
inhibitors.
That the increase in 3H during neural stimulation
was due to [3H]acetylcholine
(ACh) was confirmed by column
chromatography
(17). Radioactivity
in each 0.9-ml aliquot of
sample was calculated
as disintegrations
per minute per
square centimeter
(dpm/cm”>.
Basal release was determined
by fitting a linear regression through all basal values measured
before addition of drug. Evoked release was measured as area
under the curve. These were converted to dpm/cm2 after
comparison
with known standard
areas. Results were expressed as dpm/cm2 (17, 18).
VIP reZease studies. For studies of VIP release, the muscle
layers were removed as described (25). Three- to 5cm segments of submucosa/mucosa
were cut with a razor blade,
weighed (80-100 mg), and transferred
to a 5ml syringe. The
syringe, which was immersed
in a 37°C water bath, was
equipped with a mesh filter to prevent loss of tissue during
perfusion. One milliliter
of a modified Krebs-Ringer
solution,
which contained 0.1% gelatin, 720 U bacitracin, and 1,000 KIU
aprotinin
was perfused at a rate of 0.5 ml/min and bubbled
with 95% 02-5% CO2 (15). After an initial 20-min wash,
effluent was collected every 4 min into Eppendorf tubes kept
on ice, boiled for 10 min, and centrifuged at 3,000 g. Aliquots
(0.9 ml) of the supernatant
were stored at -70°C until
analyzed by radioimmunoassay.
In one set of tissues, basal VIP release during the first three
samples was averaged. Dimaprit
(10 I_LM) was then added,
followed by a wash period. In another set of experiments, basal
VIP release during the first three collection
periods was
followed by release during treatment of tissues with either 0.2
FM tetrodotoxin
or 0.2 PM tetrodotoxin
plus 10 FM dimaprit.
Results were expressed as a change from basal release. The
concentration
of VIP in each sample was determined
by
radioimmunoassay
(28). A 100~~1 aliquot of each sample was
tested along with VIP standards over the concentration
range
of 0.1 pM-5 mM. All samples and VIP standards were assayed
in duplicate. The VIP antiserum
(7913 from Dr. John Walsh,
UCLA) was used at a final dilution
of 1:70,000. Samples or
standards were mixed with VIP antiserum
and incubated for
24-48 h at 4°C. The following
day, 10,000 counts/min
of
12”I-labeled VIP (2,200 Ci/mol; New England Nuclear) were
added to each tube, vortexed, and incubated at 4°C. On the last
day of the assay, dextran-charcoal
was used to separate bound
lZ51-labeled VIP from free 1251-labeled VIP. The limit of
detection for the assay was 0.034 pglml. The half-maximal
inhibition
concentration
(IC& for the standard curve was
18.1 t 6.1 pg/ml. The interassay and intraassay variation was
10.3 and 7.4%, respectively.
SECRETION
Statistics. All data were expressed as means t SE; n values
reflect the number of tissues. An unpaired Student’s t-test or
analysis of variance was used to test the significance between
or among group means. A probability value < 0.05 was considered statistically
significant.
RESULTS
Effects of amiloride and N-phenylanthranilic
acid.
Amiloride (0.1 mM), which blocks epithelial sodium
channels, administered to the mucosal bath had no
significant effect on cyclical increases in I,, caused by 10
PM dimaprit (MSc: before 181 t 22 PA/cm”; after
173 t 21 PA/cm”, n = 10).
Mucosal application of the chloride channel blocker,
N-phenylanthranilic
acid (0.3 mM), significantly reduced cyclical increases in ISc evoked by 10 PM dimaprit
from control levels of 197 t 12 to 40 t 10 pA/cm2,
(n = 6, P < 0.05).
ACh release. Basal release of 3H from colonic segments was 4,812 -+ 477 dpm/cm2. Administration of 10
PM dimaprit caused a large increase in 3H of 38,493 t
5,747 dpm/cm2 (n = 7, P < 0.05). Column chromatography confirmed that the increase in 3H in response to
neural depolarization was due to [3H]ACh (17, 18). That
dimaprit depolarized submucosal neurons was reported
earlier (8). The results indicate that dimaprit caused an
increase in 3H, which is most likely to be due to an
increase in [3H]ACh.
Effect of cholinergic antagonists on dimaprit-evoked
recurrent increases in I,,. To determine whether nicotinic receptors were involved in dimaprit-evoked cholinergic transmission, one group of tissues was treated
with mecamylamine prior to administration of 10 FM
dimaprit. Mecamylamine (10 PM) significantly reduced
the amplitude of the secretory cycles (Fig. 1A). A
7.5fold higher concentration of mecamylamine (75 PM)
did not reduce further the amplitude of the secretory
cycles (Fig. 1B). To determine whether nicotinic receptors were blocked, 5 PM l,l-dimethyl-4-phenylpiperazinium iodide was added to tissues treated with 50 PM of
mecamylamine, and the secretory response was completely abolished from control levels of 322 t 48 PA/cm2
(n = 6). In the presence of mecamylamine and 0.2 PM
tetrodotoxin, secretion induced by the muscarinic agonist, bethanechol, was not altered (AZSc:40 t 8 kA/cm2,
n = 12) compared with controls (A& 38 t 9 pA/cm2),
suggesting that mecamylamine was selective for nicotinic and not muscarinic receptors.
-
400
[A
B
T
1
2
3
4
Recurrent
1
2
3
4
Cycles
Fig. 1. Effect of nicotinic
blockade
with mecamylamine
on recurrent
cyclical secretion
induced by 10 PM dimaprit
in guinea pig colon. The
peak increase in short-circuit
current
(I,,) is shown for each cycle. A:
comparison
of vehicle ( n , 12 = 5 tissues) and 10 PM mecamylamine
(CI,
IZ = 10 tissues).
B: comparison
of vehicle (D, n = 16 tissues) and 75
FM mecamylamine
( q I, n = 16 tissues).
All vehicles
are statistically
greater than mecamylamine-treated
tissues (P < 0.05).
HZ RECEPTOR-EVOKED
SECRE
G467
ION
250
2- 200
-y 150
Q
3
- 100
::
a-
50
0
0
-8
-9
-6
-7
Log [Antagonist]
M
Fig. 2. Effect of muscarinic
antagonists
on recurrent
increases
in &
induced by dimaprit
in guinea pig colon. 4-DAMP,
4-diphenylacetoxyN-methyl-piperidine
methiodide
(0); Pir, pirenzepine
(0). IZ = 4-7
tissues.
The involvement
of muscarinic
receptors in dimapritevoked recurrent
secretion was investigated
by using
several different muscarinic
receptor antagonists.
The
nonselective
muscarinic
receptor antagonist
atropine
(1 FM) decreased the amplitude of recurrent
increases
in I,, induced by 2.5 PM dimaprit from 463 t 9 to 32 t
7 pA/cm2, a reduction of nearly 97% (n = 3, P < 0.05).
Not only was the response
to dimaprit
affected by
atropine, but also the response to histamine
(115 PM)
was abolished by 5 PM atropine from control values of
269 t 37 kA/cm2 (n = 9).
Muscarinic antagonists
with a high affinity for M1 and
MS receptors
were used to determine
their ability to
reduce recurrent
increases in I,, evoked by dimaprit. At
concentrations
<25 nM, pirenzepine,
an M1 receptor
antagonist, had no effect on the amplitude of recurrent
increases in I,,. At higher concentrations,
pirenzepine
reduced the response with an I& of 200 nM. 4-Diphenylacetoxy-N-methyl-piperidine
methiodide
(4-DAMP;
M3 < M,) significantly
reduced dimaprit-evoked
response at 10 nM and higher concentrations
(Fig. 2A).
The IC&, for 4-DAMP was 11 nM.
VIP release. Basal release of VIP was 4.4 t 1.1 pg. g
wet wt1*4
min-l (n = 19) for tissues used in tetrotomin-l
doxin studies and 13 t 4 pg=g wet wt-l-4
[4CI-D-Phe’,Leu’~]VlP
400
A
B
CON/ANT
I-
cc 300
E
0
200
0
12
16
20
24
28
32
TIME
MlN
Fig. 3. Change
in vasoactive
intestinal
peptide
(VIP) release from
basal levels in the guinea pig colon. Tetrodotoxin
(0.2 PM; solid bars);
0.2 FM tetrodotoxin
plus 10 PM dimaprit
(open bars); 10 PM dimaprit
(hatched
bars). Beginning
of washout
occurred
after 20 min. IZ. = 8-12
tissues. P < 0.05 for dimaprit
at 12+ min.
of [4Cl-D-Phe6,Leu17]VIP
10 PM dimaprit.
n = 10
(n = 14) for tissues used in dimaprit studies. In the
presence of 0.2 PM tetrodotoxin, there was no significant change in VIP release from basal levels (Fig. 3, solid
bars). Addition of 10 ~.LM dimaprit to tetrodotoxintreated tissues did not cause a significant change in VIP
release from basal levels (Fig. 3, open bars). However, in
the absence of tetrodotoxin, 10 PM dimaprit evoked a
significant increase in VIP release above basal levels
(Fig. 3, hatched bars). Washout, which was begun after
20 min, caused a reduction in VIP release toward
baseline levels.
Effect of VIP antagonists. The VIP antagonist, [4Cl-IS
Phe6,Leu17]VIP, was used to determine the involvement
of VIP in dimaprit-induced recurrent increases in I,,.
The antagonist was added to the serosal bath, and 15
min later a second concentration was added, followed 15
min later by a third concentration. Both 2 and 3 FM, but
not 1 PM, [4Cl-D-Phe6,Leu17]VIP significantly reduced
the dimaprit-evoked response (Fig. 4). In another set of
tissues, a tenfold higher concentration
of [4c1-DPhe6,Leu17]VIP (30 PM) caused a further reduction to
0
-cn 100
a
a
3
MM
Fig. 4. Effect of increasing
concentrations
on recurrent
increases
in I,, induced
by
tissues. *P < 0.05.
q
s
4
2
I
0.3
MECYANT
/u M
0.3
,uM
MEUANT
3.0
/AM
Fig. 5. Effect of VIP hybrid (Hyb) antagonist
on recurrent
increases in
Is, induced by 10 PM dimaprit.
A: absence of nicotinic blockade; n = 6
tissues.
B: nicotinic
blockade
with
10 PM mecamylamine
(Met)
compared
with 0.3 PM VIP-Hyb
(n = 3-4 tissues)
on the left and
nicotinic
blockade
with 75 PM mecamylamine
compared
with 3 PM
VIP-Hyb
(n = 7 tissues) on the right. Solid bars, vehicle treated;
open
bars, VIP-Hyb
antagonist
(Ant). Con, control.
*P < 0.05.
G468
H2 RECEPTOR-EVOKED
SECRETION
SECRETOMOTOR
A
INTERNEURONS
EplTHFl
IUM
TERNEURON
EPITHELIUM
SECRE
NEURONS
*ii
v
Fig. 6. Model to illustrate
2 possible neural circuits
involved
in dimaprit-evoked
recurrent
secretion.
A: dual input
from interneurons
onto a cholinergic
secretomotor
neuron.
Dimaprit
activates
Hz receptors
on cholinergic
and
VIP-e@
interneurons
that are synaptically
coupled to cholinergic
secretomotor
neurons.
Atropine
(Atr) will block
secretion
by acting at epithelial
muscarinic
receptors
(M). Mecamylamine
(Met)
will reduce recurrent
secretion
by
50% by blocking
nicotinic
(N) transmission
from the cholinergic
interneuron
to the secretomotor
neuron.
VIP
antagonists
(VIP/Ant)
will reduce recurrent
secretion by preventing
transmission
from the VIP-ergic
interneuron
to
the cholinergic
secretomotor
neuron.
B: input from a single cholinergic
interneuron
onto 2 secretomotor
neurons.
Dimaprit
activates
H:! receptors
on cholinergic
interneurons
that release acetylcholine
at N and M synapses with
choline@
and VIP-e@
secretomotor
neurons,
respectively.
Recurrent
cycles of secretion
will be abolished
by Atr
acting at both neural and epithelial
M receptors.
Blockade
of N receptors
will reduce recurrent
secretion
by half and
will be further
reduced by VIP antagonists
(VIP/ Ant) acting at epithelial
receptors.
77 t 32 PA/cm2 from control values of 316 t 30 PA/cm2
(n = 6).
To verify that this antagonist
was specific for VIP
receptors,
10 PM atropine was added, and the neurons
were stimulated by an electrical field in the presence and
absence of [4cl-D-Phe6,Leu17]VIP.
This noncholinergic
component of neurally evoked chloride secretion is due
to VIP (25). At concentrations
of 10 and 30 PM, the VIP
antagonist
reduced noncholinergically
evoked increase
in ZSc and chloride secretion due to release of VIP in
response to electrical field stimulation
(stimulus parameters: 2 Hz, 25 V, 0.5 ms) by 21 t 5 and 45 t 5%
b-8 = 5-9), respectively,
compared with vehicle-treated
controls (- 3 t 5950, 7 t 970, n = S-10). The ability of
the antagonist to attenuate this response indicates that
the antagonist is specific for VIP.
Dimaprit
(10 PM) evoked recurrent
increases in ZSc
that were significantly
reduced by 0.3 PM VIP hybrid
(VIP-Hyb)
antagonist
(Fig. 5A). When 10 PM of mecamylamine was added to prevent nicotinic transmission
from choline@
interneurons,
there was a reduction in
recurrent increases in I,, as seen earlier (Figs. 1 and 5B).
Even though nicotinic transmission
had been abolished,
the VIP-Hyb
antagonist caused a concentration-dependent decrease in the response to dimaprit (Fig. 5B).
DISCUSSION
The results indicate that blockade of chloride channels, but not epithelial sodium channels, reduced the
amplitude of recurrent
increases in I,,. This observation
provides additional evidence that dimaprit-evoked
increases in I,, reflect chloride secretion without
affecting
sodium absorption.
They extend previous findings that
blockers
of the sodium-chloride-potassium
cotransporter, which is necessary for chloride secretion, and
chloride-free
solutions reduced recurrent increases in I,,
(29). Taken together, the evidence supports the conclusion that dimaprit
causes recurrent
cyclical chloride
secretion.
The present study gives insights
into the neural
pathways that mediate dimaprit-evoked
recurrent cycles
of secretion when H2 receptors are activated. The results
provide direct evidence for involvement
of choline@
neurons, because dimaprit caused a large increase in
[3H]ACh release above basal levels of 3H. Furthermore,
the involvement
of cholinergic neurons is further substantiated
by pharmacological
blockade by choline@
antagonists.
This observation
is consistent
with previous findings in bovine milk-sensitized
animals (16, 18).
Choline@
interneurons
were implicated by the finding
Hz RECEPTOR-EVOKED
that nicotinic blockade reduced the amplitude of recurrent secretion by - 50%. Since a maximal concentration
of mecamylamine
did not abolish cyclical secretion, the
remainder
of the response must have resulted from
dimaprit’s
activation of neurons with nonnicotinic
synapses. This conclusion is based on the assumption
that
75 PM mecamylamine
provided complete blockade of
nicotinic receptors and is probably valid, because a lower
concentration
of mecamylamine
also reduced secretion
by an equivalent amount.
Choline@
neurons, which release ACh at muscarinic synapses, were also involved in recurrent secretion.
In the guinea pig colon, the antagonist
with a high
affinity for M3 receptors,
4-DAMP,
was more potent
based on I& values than was pirenzepine, which has a
high affinity for M1 receptors (5, 24, 26). This observation is consistent with previous reports from this laboratory showing
that 4-DAMP
was more potent than
pirenzepine
in reducing chloride secretion evoked by
electrical field stimulation
of submucosal
neurons and
in inhibiting
[3H]quinuclidinyl
benzilate binding from
mucosal scrapings (2 1).
VIP-e@
neurons
are also involved in dimapritevoked recurrent
secretion. Evidence for this conclusion
comes from the finding that dimaprit evoked a tetrodotoxin-dependent
release of VIP. Additional evidence for
involvement of VIP-e@
neurons comes from the observation that recurrent
secretion
was significantly
reduced by two different VIP antagonists
(13, 23). [4c1-DPhe6,Leu17]VIP
not only inhibited
dimaprit-evoked
secretion but also inhibited electrically evoked noncholinergic secretion, which is mediated by VIP-e@
neurons (25). Specificity of the antagonists
for VIP receptors is further evidenced by the observation that one or
both of these antagonists reduced VIP-evoked depolarization in submucosal neurons (unpublished
observations)
and inhibited VIP-induced
chloride secretion in colonic
epithelial cells (23).
Two working
models of the neural circuitry
controlling the epithelium
can explain most of the results.
These models take into account the fact that activation
of muscarinic
receptors
or VIP receptors
either on
neurons or on epithelial cells caused colonic secretion in
guinea pig colon (20, 21, 25). The finding that muscarinic blockade abolished dimaprit-evoked
recurrent secretion suggested that either 1) choline@
secretomotor
neurons, which transmit
signals to the epithelial cells
via muscarinic receptors, are the final common pathway
to the epithelium (Fig. 6A) or 2) choline@
interneurons are synaptically
coupled to choline@
and noncholine@
secretomotor
neurons via nicotinic and muscarinic synapses, respectively
(Fig. 6B). In this latter case,
blockade of muscarinic
receptors would be expected to
prevent recurrent
secretion by blocking ganglionic transmission to noncholinergic
secretomotor
neurons and by
inhibiting
neuroepithelial
transmission
from cholinergic secretomotor
neurons (Fig. 6B).
The results implicate VIP-e@
neurons in dimapritevoked recurrent
secretion, and these could be either
VIP-e@
interneurons
(Fig. 6A) or VIP-e@
secretomotor neurons
(Fig. 6B). VIP antagonists
could reduce
G469
SECRETION
recurrent
secretion by blocking VIP receptors on epithelial cells and thereby preventing transmission
via VIPergic secretomotor
neurons at neuroepithelial
junctions
(Fig. 6B). This is certainly
a possibility,
since VIP
receptors have been detected on colonocytes in a previous study (25). The results, however, cannot distinguish
between this possibility and the possibility that the VIP
antagonists
decreased recurrent
cyclical secretion
by
inhibiting ganglionic transmission
when they bound to
neural VIP receptors (Fig. 6A). VIP receptors are present on submucosal neurons as evidenced by the finding
that exogenous VIP caused long-lasting
depolarization,
which was attenuated by VIP antagonists
(unpublished
observations;
Ref. 4). That these neurons were involved
in secretion is likely, because, in the presence of neurogenie tone, VIP-evoked
secretion was tetrodotoxin
sensitive (20,25).
A further consideration
in developing a working model
was the observation
that a VIP antagonist
suppressed
recurrent
secretion that was resistant to nicotinic blockade. This implied that VIP release was not dependent on
signals from choline@
interneurons
via nicotinic synapses (Fig. 6, A and B). The results are consistent with
H2 receptors
present on VIP-e@
interneurons
(Fig.
6A) or on choline@
interneurons
synaptically
coupled
via muscarinic
synapses to VIP-e@
secretomotor
neurons (Fig. 6B).
We have previously demonstrated
in the bovine milksensitized
guinea pig model that mast cells release
histamine and other inflammatory
mediators. Release of
mast cell products causes recurrent
cycles of secretion
that are neurally mediated and sensitive to histamine Hz
antagonists
(16, 18). The findings in the current study
underscore
the importance
of both cholinergic
and
VIP-e@
pathways
in controlling
ion transport
in response to the activation of histamine H2 receptors, and
they provide new insights into how signals from the
immune system can modulate activity of the enteric
nervous system.
Appreciation
is expressed
to Dr. J. Grider
for advice with the VIP
radioimmunoassay,
to Dr. A. Kuwahara
and J.-Y. Cho for assistance
with some of the studies, to Yun Xia (unpublished
observations),
and
to Paula Fox and Maia Bailey for technical
assistance.
The project was supported
by National
Institute
of Diabetes
and
Digestive
and Kidney Diseases Grant ROl DK-37240.
Address for reprint
requests:
H. J. Cooke, Dept. of Physiology,
1645
Neil Ave., The Ohio State Univ., Columbus,
OH 43210.
Received
15 November
1993;
accepted
in final
form
3 October
1994.
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